Container for transporting bacterial cultures.



G. H. EARP-THOMAS.

CONTAINER FOR TRANSPORTING BACTERIAL CULTURES. APPLICATION FILED JAN.15. 1909. RENEWED AUG. 31. 1914.

Patented Apr. 27', 1915.

riou's sterilized and other GEORGE H. E ias-THOMAS, or

GLEN RIDGE, NEW JEnsEv.

I CONTAINER FOR TRANSPORTING IBACTERIAL CULTURES.

" Specification of Letters Patent.

Patented Apr. 27-,1915.

Application filed January 15, 1909, Serial No. 472,476. Renewed August31', 1914. Serial No. 859,565.

To all whom it may concern: 1

Be it known-that I, GEORGE H. EARP- THOMAS, subject of the King of GreatBritain, residing at Glen Ridge, l'ilssex'county, New Jersey, haveinvented certain new and useful Improvements in Containers forTransporting Bacterial Cultures; and I do hereby declare the followingto be a full, clear, and exact description of the invention, such aswill enable others skilledv in the art to'which it appertains to makeand use the same. i

This invention relates to-me'ans for permitting the passage of air orother gas between the interior of closed containers or other .vesselsand the external air or.gas, while at the same time all bacterial andother contaminations are excluded from the interior ofthe container, andthe escape'of liquids fro-1n or the entrance of liquid into thecontainer is prevented, and loss of moisture. from the container byevaporation is reduced tosu'ch an extent as to be prac ticallyinappreciable.

The invention may be employed for preserving in an uncontaminatedcondition vasubstances to which it is necessary or advisable to supplyair or other gases, and is of especial value in preserving suchsubstances during transe portation and storing in suitable containersunder the conditions of commercial distribution. Eihe invention willalso be found adaptable for use in connection With .or when applied tocontainers for various volatile drugs and chemicals. y i

The" invention has been made, however,

, especially with the idea of providing, and

therefore, to the preservation of micro-' organisms, and it comprisesatransportable or dlstributing package of micro-organisms, such package.COIIiPIlSlIlg a nutrit ve mediam inoculated with the desired organismsand contained in a vessel having means such as above referred toaffording communication between the interior of the vessel and theexternal airor gas and protecting the contents of the vessel fromcontamina,- tion and restricting loss by evaporation and preventing lossof liquid from the vessel, all as more fully hereinafter set forth andas claimed.

The commercial production and distribution of cultures of variousbacteria and the like has become desirable and necessary in recentyears, but methods and means hereto-' fore suggested to effect suchdistribution have been unsatisfactory for the reason,

but if the vessel containing the culture is hermetically sealed then theperiod for which the culture will remain in good condition will belimited by the amount of air or gas contained in the vessel. To avoidthis difliculty, it has been a custom followed to a considerable extentto distribute various aerobic bacteria in containers, usually of glass,having the neck ormouth of the container stoppered with a plug of cottonor similar fiber which serves as an air filter to prevent contaminationby organisms carried in the air and dust while permltting the necessaryaccess of air to the bacteria in the container. 1 Such a device servesvery Wellin the laboratoryvwhere the container can be kept standingupright and the cotton plug kept dry, but it is not satisfactoryunder'the conditions of commercial transporamong others,that manydifliculties attendtation where inversion of the container can hardly beprevented. If the plug becomes moist, as is 'apt to happen; when thecontainer is inverted and the culture medium or liquid separatedtherefrom comes in contact with the cotton, the organisms which havesettled on it from the air. can readily grow by simple longitudinalgrowth and sub-division until they ultimately attain the other side ofthe cotton plug and fall or are projected into and contaminate theculture within the container.

Another ojection to the use of ordinary cott0nplugged containers fordistributing cultures is that the cultures suffer a very considerableloss of moisture from evaporation, which in a comparatively short timechanges the character of the medium, not only with respect to itsmoisture content but with respect to its saline content. The ordinarycotton plugged container is equally objectionable for gas-producingbacteria, and a hermetically sealed container is not suitable for thembecause of the pressure which the gases they produce might developwithin the container.

In the present invention I have devised a simple, cheap and eflicientmeans for securing all the advantages of both the hermetically sealedand the cotton-plugged culture containers, while avoiding theobjectionable features of and difficulties attendant upon both. Suchmeans comprises a gas conduit or tube for connecting the in terior of anotherwise hermetically sealed container with the external air or gas,which conduit or tube may be of comparatively small cross section andcontains means for substantially preventing passage of microorganismsthrough said conduit in either direction, such means comprising in thepreferrcd form aplurality of separate air filters therein formed ofplugs of cotton, silk fiber, asbestos or other suitable fiber, butpreferably of non-absorbent cotton, and preferably spaced away from eachother by air spaces, such filters or fiber plugs being properlycompacted. WVith this device, even though an end plug in the tube orconduit should become wet, a plug beyond the wet plug will remain dryand will protect the contents of the container against contami nation,while the passage of air or other gas through the'conduit will bepermitted even if the end plugs are wet. Three or more such spaced airfilters or fiber plugs are preferably placed in the conduit, since whenat least three such filters are used there will be at least one whichwill be protected by the filters beyond it from access of moisture fromeither the interior or ,the exterior of the container. More than threeplugs may be used, however, and, on the other hand, the use of twoseparated plugs will be found eflicient for many purposes, and a singlecotton plug may be used as hereinafter explalned in connection withcertain other features of the invention with good results.

The conduit containing the separated air filters is preferably arrangedso that it extends within the container and is open to the interior ofthe container at a point removed from any wall thereof, so that with thecontainer only partially filled liquid cannot gain access to theconduit, or cannot gain access in any material amount, whatever theposition of the container. The opening from the conduit into thecontainer is preferably also comparatively small, thereby furtherminimizing loss of moisture by evaporation and lessening the liabilityof liquid entering the conduit.

'A container having a gas conduit provided with a plurality of spacedair filters such as above described and containing a nitrogen-freenutrient medium inoculated with nitrogen-gathering bacteria of theleguminous plants forms a desirable type of culture for commercialdistribution, the medium being protected against contamination byforeign organisms and from loss of moisture, while the bacteria remainin an active, virulent condition for long periods of time. Thepreparation of such inoculated culture media is described in my priorUnited States Patents 816,850, datedrApril 3, l79O6, and 865,965, datedSeptember 10, 190

The legume bacteria require nitrogen and in order to be preserved in anactive and virulent condition in a nitrogen-free medium such as thedescribed medium they must be able to obtain nitrogen from theatmosphere within the container. For long preservation of a culture ofthese bacteria in a nitrogen-free medium, therefore, access of air tothe interior of the container is necessary unless the container is madeso large relatively to the amount of inoculated medium which it containsthat the supply of air within the container will be sufficient for thetime for which it is desired to preserve the culture. Practically,therefore, it is necessary to provide for access of air if the cultureis to remain in an'active, virulent condition for a long period of time.

The present invention provides for such access of air while protectingthe contents of the container from contamination and from change due toloss of moisture by evaporation.

The use of a gas conduit providing communication between the atmospherewithin a container and the external atmosphere through a restrictedopening and a dry cotton plug is also of great value in the preparationand keeping of sterilized medium until wanted for inoculating.

In the accompanying drawings I have shown more 01' less diagrammaticallycertain embodiments of the invention.

In said drawing: Figure 1 is a vertical section of a distributingpackage embodying the invention. Fig. 2 isa similar view showing aslightly modified construction. Fig. 3 is a broken view of a container,showing a stopper constructed in accordance with the invention andprovided with a *"dust cap, and showing a slightly different form ofconduit tube. Fig. 4 is a similar view showing a still further modifiedform of conduit tube.

Referring to the drawings, and first to Fig.1, 1 is a container orreceptacle carrying'a body 2 of nutrient mediumcontaining the desiredorganisms, as, for example, a body of a jelly-like medium containingnodule-forming nitrogen-fixing bacteria intended for inoculating cloveror other legu'mes. As shown, the receptacle is of glass and is bottleshaped, though it might have other shapes or be of other material, andis provided with the perforated stopper 3 which may be of rubbercomposition or other suitable material. Through this stopper extends agas conduit tube 4 ending above or flush with the top of the stopperand, as shown, freely open to the external atmosphere. The conduit tubemay be of glass or aluminum or other suitable material. In the tube area plurality of air filters 5, advantageously three orfi more, which maybe of cotton, silk, flax, asbestos or kither suitable fiber. The fiberplugs forming the air filters are preferably spaced apart and separatedby distinct air spaces 6 as shown. Much of the advantage of the separateplugs may be secured, however by placing the plugs so close togetherthat the more loosely compacted end portions of contiguous plugs will bein contact,'and such a disposition of the fiber plugs might in somecases be employed. The conduit tube as shown extends down to about themiddle of the container above normal level of the body of nutrientmedium therein. It is formed as shown in this figure with a reduced end7 bent into a curve. As an aid to placing the plugs of cotton or otherfiber forming the air filters in the conduit tube, it is convenient touse short spacing pieces 8 between the several filters or fiber plugs inthe tube, such spacing pieces being formed of short lengths of glasstubing, short glass rods of smaller cross section than the interior ofthe conduit tube, or of pieces of other form. The advantage of havingthe reduced end 7 of the conduit tube bent around into circular form, asshown in this figure, is that the chance I that a drop of liquid eventemporarily entering or closing the end of the conduit or tube isthereby very largely decreased, especially when the end of the tube isdrawn out or otherwise reduced in size. The advantage of the orifice atthe end of the tube being smaller than the passage through the body ofthe tube is that evaporation through the tube is thereby largelydecreased and the chance of entrance of liquid into the tube alsoconsiderably lessened.

A container open to the atmosphere and provided with a plurality ofspaced fiber plugs or air filters, as shown in Fig. 1, is eminentlyadapted for the transportation of media containing the nitrogen-fixingbacteria of the legumes, since it provides for the necessary supply ofair to the interior of the container while protecting the inoculatedmedia from contamination and preventing loss of liquid and largelyreducing evaporation from the container, as hereinbefore pointed out.The inoculated nutrient me dium within the container may be any mediumsuitable for the growth of such nitrogen-fining organisms and may beeither liq uid or solid. If liquid, its amount should, of course, not begreat enough to cause sub-v mersion of the orifice of the conduit. If asolid jelly medium be used, the mass, similarly, should not reach theorifice. When the organisms are carried on moistened solids, such asthreads, potato fragments and the like, the bulk of the-medium is not soimportant since ordinarily not nough moisture will form to reach thehrifice of the conduit nomatter what the position of the container. lhestructure shown iii-Fig. 1 may, of course, be employed fortransportation of micro-organisms other than the nitrogenfixing bacteriaof the legumes, the medium employed. being, of course, in all cases suchas is suitable" to the organisms to be transported. So also thecontainer shown with its air conduit provided witha plurality of cottonplugs or air filters may be employed for the transportation andpreservation of other substances, as hereinbefore pointed out. i

In making up the complete transportable package, including thecontainer, theconduit tube with its fiber plugs or air filters,

and the inoculated medium, the desired amount-off the medium may beplaced in the container and the stopper carrying the conduit tube withits air filters then placed securely in the neck of the container. Thecontainer with its assembled stopper, conduit tube and medium are thensterilized in any suitable way, as by streaming steam; or

in the autoclave. It is possible to thus steriappreciable loss ofmoisture than when the ordinary cotton plug stopper is used. if themedium is to be kept uninoculated for a very long period aftersterilization, the open end of the conduit tube may be sealed withremoved when it is desired to inoculate the medium. I In order toinoculate the medlum, it is only necessary to carefully remove thestopper carrying the conduit tube, then inoculate the medium with theordinary inoculating needle or otherwise, and replace the stopper aftercarefully flaming it, which may thereafter remain in place until it isdesired to make use of the culture. The container will then be kept at atemperature a suitable for the development of the organisms with whichthe medium has been inoculated until the desired growth has resulted,after which it may be kept for a long period of time and transported asmay be desired, with the certainty that the organisms and medium withinthe container will remain free from contaminations, and that theorganisms will remain in a living, virulent condition.

In placing the several cotton plugs in the conduit tube, the first plugis pressed toward the inner end of the tube but preferably not into thereduced portion of the tube adjacent t9 its inner end, and the remainingplugs "are then placed successively in the tube and pushed into theirrespective positions so as to leave a perceptible air space between eachsuccessive plug. The object of not having the inner plug extend into thereduced portion of the tube is to lessen the liability of such plugbecoming moistened by any liquid which may enter the end of the tube. Itmay be desirable in some cases to treat one or more of the cotton plugswith a suitable antiseptic, such as corrosive sublimate, 1 to 1000 beinga suitable strength, but the first plug at the inner end of the conduittube should, however, not be so treatedas it is not desirable that anyantiseptic be allowed to reach the interior of'the container.

The structure shown in Fig. 2 is similar to that of Fig. 1 save that thecurve on the reduced end of the conduit tube is omitted, and that a gasholder 9 is provided, to the interior of which the outer end of theconduit opens, the reduced portion of the conduit serving, in thisspecific embodiment of the invention, as means to prevent liquid fromentering the conduit tube and reaching the plug of fibrous material.Such gas holder may be a simple gas bag provided preferably with aninlet 10 and may be used for holding any gas which it is desired tosupply to the organisms or substance within the container. Such a gasholder might also be used for determining the amount of air, nitrogen orother gas absorbed or made use of by certain organisms within thecontainer.

The reduced end portion of the conduit tube may vary in size and lengthaccording to the necessities of any particular case. In

some cases and more especially for use with liquids, it is desirable tohave the end portion of the conduit tube drawn out to such a small sizeas to form a capillary tube. It is also desirable for use with liquidsthat the extreme end of the conduit tube be formed with a fine point anda very small orifice, so that a drop of liquid will not be sustainedthereon and the possibility of liquid entering the tube will be almostentirely eliminated. The limitation as to the fineness of the point willbe found in the capacity of the orifice at the point for the passage ofair or other gas, and this limitation will be governed by the airrequirements of the vessel dependent upon its size and the necessitiesof its contents. reduced and pointed end is shown on the conduit tube inFig. 2. It will be understood, however, that this pointed end withanextremely reduced orifice might be provided on a conduit tube having acurved end portion, as shown in Fig. 1, or on a tube of Such a end ofthe tube is, however, cut or finished at an angle so that any liquidwhich may collect on the tube will not be aptto close the orifice at theend of the tube. This figure shows further the stopper as provided withan annular flange or shield 11 which when the stopper is in position inthe neck of the container extends over the top of the neck of thecontainer to cover and shield .the same from micro-organisms, dust, etc,

which might otherwise settle thereon and might possibly enter the neckof the bottle if the stopper were removed. Such a shield carried by thestopper will be found valuable where a container provided with a stoppercarrying the air conduit with its plurality of air filters is employedfor laboratory or other use which requires the more or less frequentremoval and replacing of the stopper.

Fig. 4 shows a still further variation of the conduit tube in which theextreme inner endof the tube is closed and an opening to the interior ofthe container is formed by heating the side of the tube near its innerend and blowing out a comparatively small opening as shown at 12 in thisfigure. This figure shows also a cap fastened over the top of thestopper and neck of the receptacle. Such a cap might be for the purposeof protecting the stopper and neck of the bottle, or for holding thestopper in place,

. water from reaching the first fiber plug in V the conduit tube, and insuch case a conduit pass, or it may be suitably constructed or,

arranged to permit air to have access to the conduit tube. A cap ofpaper parchment or other suitable material would serve to prevent, underordinary circumstances at least,

tube with only two filter plugs, as shown in this figure, would make anefficient device. In fact, as the chance of liquid reaching the lugnearest the inner end ofthe tube is ut slight, especially when a solidor semisolid medium or other substance is in the bottle, a single plugwill in many cases answer to prevent the entrance of contaminations. If,however, only one plug were employed, it would be preferable to use atube having its inner end formed to avoid liquid entering through theorifice and reaching the plug, as for example the curved reduced endshown in Fig. 1, or a capillary tube ending in a fine orifice as shownin Fig. 2. f

It is preferable to make the container, stopper and conduit tube ofseparate parts was shown for economy and ease in assemblage, rather thanto make an intergral structure as by sealing a glass conduit tubethrough one of the walls of the glass container. A further advantage ofthe arrangement shown is that the stopper when of proper material suchas rubber or a suitable composition acts as a cushion for the conduittube andavoids danger of breakage of the latter. The whole device mightbe made of metal, in which case, of course, the objection offrangibility would not obtain as in the case of an integral glassstructure.

Another advantage of the arrangement shown is that the stopper andconduit tube may be used for different containers or vessels, just as anordinary rubber or cork stopper may be used. The conduit tube might, ofcourse enter the vessel at any point and might for some purposes be. arranged to extend partlyor mostly outside of the container or vessel, butthe arrangement shown, in which the opening from the conduit into theinterior of the container is located near the center of the container,

is preferable for most purposes and especially where the container is tobe used for transportation or is otherwise liable to inversionf What isclaimed is:

1. A container comprising a closed receptacle and a conduitcommunicating with the atmosphere within the receptacle and containingthree or more separate plugs of fibrous material.

2. A container comprising a closed receptacle and a conduitcommunicating with the atmosphere within the receptacle at a pointremoved from the walls thereof, said conduit containing a plurality ofseparate plugs of fibrous material and the inner end of said conduitbeing bent in circular form and ending at such point of communication.

8. A container comprising a closed receptacle, a conduit tube connectingthe atmosphere Within the receptacle with the atmosphere external to thereceptacle and having its inner end reduced and curved circularly andending and open to the interior of the receptacle at a point removedfrom the walls of the receptacle, and a plug of fibrous material in theconduit tube.

4. A container comprising a receptacle, a stopper for closing the mouthof the receptacle, a conduit tube extending through the stopper and opento the interior of thereceptacle at a point removed from the wallsthereof, a plug of fibrous material in the conduit tube, and means forpreventing liquid entering the conduit tube and reaching the plug offibrous material.

5. A container adapted for transporting and preserving aerobic bacterialcultures comprising a receptacle, a stopper for closin the mouth of thereceptacle, a conduit tu e traversable by air extending through thestopper and open to the interior of the receptacle at a point removedfrom the walls thereof, said conduit tube containing a plurality ofseparate plugs of fibrous material and having a reduced end throughwhich 1 connection is established with the interior of the receptacle atsaid point removed from the walls of the receptacle.

6. A container adapted for transporting and preserving aerobic bacterialcultures 105 comprising a receptacle, a stopper of flexible and elasticmaterial for closing the mouth of the receptacle, and a conduit tube ofglass open to the exterior of the receptacle and extending through thestopper and open to 11 the interior of the receptacle at a point removedfrom the walls thereof, said conduit tube containing a plurality ofseparate plugs of fibrous material.

7. A container adapted for transporting 11 and preserving aerobicbacterial cultures comprising a receptacle, a stopper for closing themouth of the receptacle, and a conduit tube extending through thestopper and open to the interior of the receptacle 120 at a pointremoved from the Walls thereof and connecting the atmosphere within thereceptacle with atmosphere external thereto, said conduit tube beingprovided with means for intercepting micro-organisms passing 1therethrough and preventing such microorganisms from entering thereceptacle.

8. A container ada ted for transporting and preserving aero ic bacterialcultures comprising a receptacle, a stopper for clos- 130 ing the mouthof the receptacle, a conduit tube extending through the stopper and opento the interior of the receptacle at a point removed from the wallsthereof and connecting the atmosphere within the receptacle withatmosphere external thereto, said conduit tube being providedwith meansfor intercepting micro-organisms passing therethrough and preventingsuch micro-organisms from entering the receptacle, and means forpreventing the entrance of liquid from the receptacle into said conduittube.

9. A container comprisingv a receptacle, a stopper for closing the mouthof the receptacle, and a conduit tube extending through the stopper andopen to the inter1or of the receptacle at a point removed from the wallsthereof and connecting the atmosphere within the receptacle withatmosphere external thereto, said conduit tube being provided with meansfor intercepting mlcro-organisms passing therethrough and having itsinner end reduced and bent in such manner as bto hinder entry of liquidinto the conduit tu e.

10. A container adapted for transporting.

tube extending therethrough and prolonged for a distance beyond theinner end of said stopper body, said conduit tube containing three ormore separate plugs of fibrous ma terial.

12. A stopper for containers or rece tacles comprising a stopper body ofrub er or other suitable flexible and elastic material, and a conduittube extending through the stopper body and prolonged for a distancebeyond the inner end of said stopper body, said conduit tube containinga plurality of separate plugs of fibrous material and having its endwhich is intended to extend into the container or receptacle reduced andbent into a curve to hinder entry of liquid into the conduit tube.

13. A stopper for containers or receptacles adapted for transporting andpreserving aerobic bacterial cultures comprising a stopper body ofrubber or other suitable flexible and elastic material, and a conduittube of glass extending therethrough and prolonged for a distancebeyond'the inner end of the stopper body, said conduit tube beingtraversable by air and containing a plurality of separate plugs offibrous material.

14. A stopper for containers or receptacles adapted for transporting andpreserving aerobic bacterial cultures comprising a stopper body ofrubber or other suitable flexible and elastic material, a conduit tubetraversable by air extending therethrough and prolonged for a distancebeyond the inner end of said stopper body and containing a plug offibrous material, and means for preventing liquid enterin the conduittube and reaching the plug of fibrous material.

15. A stopper for containers or receptacles adapted for transporting andpreserving aerobic bacterial cultures comprising a stopper body ofrubber or other suitable flexible and elastic material, and aconduittube traversable extending therethrough and prolonged for adistance beyond the inner end of the stopper body, said conduit tubebeing provided with means for intercepting microorganisms passingthrough the tube and for retaining the same within the tube.

' 16. Astopper for containers or rece tacles comprising a stopper bodyof rub er or other suitable flexible and elastic material,

and a conduit tube extending therethrough and prolonged for a distancebeyond the innerend of the stopper body, said conduit tube beingprovided with means for intercepting micro-organisms passing throughthetube and for retaining the same within the tube and having its endbeyond the inner end of the stopper body reduced and bent into a curveapproximating nearly a complete circle. 1

17. A container adapted for transporting and preserving aerobicbacterial cultures which comprises the combination, with a closedreceptacle, of a conduit tube supported in such manner as tobe locatedsubstantially wholly within said-receptacle and aliording an air passageextending from the exterior of the receptacle to the interior thereof ata point removed from the receptacle walls, and separate plugs of fibrousmaterial disposed in said tube to prevent passage of micro-organismstherethrough.

18. A container adapted for transporting and preserving aerobicbacterial cultures which comprises the combination, with a closedreceptacle, of a conduit tube sup ported in such manner as to be locatedsubstantially Wholly Within said receptacle and affording an air passageextending from the exterior of the receptacle to the interior thereof ata point removed from the receptacle walls, and means in said tubetoprevent passage of micro-organisms therethrough,

rovision being also made to hinder entry of liquid from the receptacleinto said tube. a

19. A gas filtering device formed of a conduit tube containing aplurality of separate plugs of fibrous material and having one endreduced and bent into a curve to hinder entry of liquid into saidconduit tube.

20. A bacterial distributing package commenace prising a closedreceptacle, a moist culture medium carrying a culture of micro-organismscontained in the receptacle, and a gas conduit communicating-With theatmosphere within the receptacle at a point removed from the wallsthereof, said. conduit being provided 'with means for interceptingmicroor anisms passing therethrough, and said cu turemedium being inamount insuflicient to submerge the orifice of the conduit inanyposition of the receptacle.

21. A bacterial distributing package com prising a closed receptacle, amoist culture medium carrying a culture of nitrogen-fix- I ing organismscontained in the receptacle,

and a conduit leading to the interior of the receptacle and having anorifice located at fibrous material forming a gas filter, and

said culture medium being in amount insufficient to submerge the orificeof the conduit in any position of-the receptacle.

In testimony whereof I have hereunto afiixed my signature in thepresence of two witnesses.

GEORGE H. EARP-THOMAS. Witnesses HARRY 'S. CoNnoLLY, A. L. KENT.

